Purification and activity assurance of precipitated heterologous proteins

ABSTRACT

A heterologous expression product in host cells is separated from the host cell material by suspending the material in a buffered solution, disrupting the cells and separating the product as refractile material. The refractile material can be dissolved in a strongly denaturing solution, which is then weakened while keeping the protein in solution, thereby allowing unfolding and refolding of the protein.

This application is a continuation of application Ser. No. 452,187,filed Dec. 22, 1982, now abandoned.

BACKGROUND OF THE INVENTION

Recombinant DNA technology has permitted the expression of exogenous orforeign (heterologous) proteins in bacteria and other host cells. Undersome conditions, and for some proteins, these heterologous proteins areprecipitated within the cell as "refractile" bodies. The presentapplication concerns procedures for recovering these heterologousproteins and for restoring them, if necessary, to their active forms.

A large number of human, mammalian, and other proteins. including, forexample, human growth hormone, (hGH) bovine growth hormone (bGH) and anumber of interferons have been produced in host cells by transfectingsuch cells with DNA encoding these proteins and growing resulting cellsunder conditions favorable to the expression of the new heterologousprotein. Viral coat proteins, such as capsid proteins of foot and mouthdisease (FMD) virus and the surface antigenic protein of hepatitis Bvirus (HBsAg) are still other examples of heterologous proteins whichhave also been produced in suitable recombinant DNA engineered hosts.The heterologous protein is frequently precipitated inside the cell, andconstitutes a significant portion of the total cell protein.

In a large number of important cases, such as those of hGH, porcinegrowth hormone (pGH), bGH, FMD, and fibroblast interferon (FIF), it hasbeen observed that the heterologous proteins produced are not onlypresent in large quantity, but are precipitated within the cell in theform of "refractile" bodies. The term "refractile" is used because thesebodies can actually be seen using a phase contrast microscope. Undermagnifications as low as 1000 fold, these precipitated protein bodiesappear as bright spots visible within the enclosure of the cell.

Recovery of the desired protein which is in the form of such refractilebodies has presented a number of problems. First there is the obviousneed to separate the refractile protein, which is encased within thecell, from the cellular material and proteins harboring it. Second, itappears that while the refractile body may often consist of a largepercentage of the desired heterologous protein, and only a small portionof undesired ones, in some instances there are sufficient proteincontaminants that these must be removed to isolate desired polypeptidesequence. Third, and perhaps most troublesome, the refractile bodyprotein is often in a form which, while identifiable as the desiredprotein, is not biologically active. It is believed that this inactivityis due to incorrect folding or conformation of the heterologous proteineither before or after intracellular precipitation, or during theisolation process.

It has now been found that these problems can be overcome by utilizingprocedures which, in their various aspects, succeed in removing thecontaminating host cellular protein, solubilizing the precipitatedrefractile protein, and restoring the heterologous protein to a formwhich is active in biological assays.

SUMMARY OF THE INVENTION

This application is directed to various aspects of an invention whichprovides an overall solution to what has emerged as a generalizedproblem:--recovering, in active form, proteins which have been producedin host cells, which are heterologous thereto, and which are at leastpartially deposited inside the cells as refractile bodies i.e. clumps ofinsoluble protein. This approach, which provides an effective protocolfor recovery of heterologous proteins from cell cultures whereinrefractile bodies are formed, is presented in diagramatic form,including the various available alternatives, hereinbelow as Scheme 1.

Briefly described, scheme 1 has several phases: First, the precipitatedinsoluble protein is liberated from the cells by employing means whichdisrupt the outer cell wall/membrane under conditions comprisingsufficient ionic strength and proper pH so that the host cell proteins,provided the cells are sufficiently disrupted, will be solubilized, orat least will fail to be brought down by low speed centrifugation.Accordingly, upon centrifugation the desired refractile protein will beaccumulated in the pellet, and most of the contaminating proteins willthen remain in the supernatant. The pellet, however, may containcontaminating proteins for several reasons. First, the originalrefractile body may not have been entirely comprised of the desiredprotein. Second, fragments of cell walls or membranes may beinsufficiently disrupted so that they remain with the pellet and areundetected even upon microscopic examination of the pellet. However, thepellet which results will be predominantly the desired protein, and,unlike the situation found in standard protein purification proceduresused in enzymological studies, the problem becomes one of removingcontaminants from a basically pure product, rather than isolating asmall component of a complex mixture.

(In some instances, notably that of human growth hormone, theheterologous protein produced by the bacterium or other host organism isonly partially in refractile form as the cells are grown and the genefor the protein expressed. In those instances, it has been found thatenhancement of the quantity of the desired protein contained in thepellet can be obtained by treating the cells, prior to disruption, withmethods which were traditionally designed to kill the cells incompliance with safety precautions related to recombinant DNAtransformed cells. Such techniques as acid, heat, or treatment withnonpolar solvents, appear to complete insolubilization of partiallysoluble proteins.)

Having secured a preparation which is predominantly the desired protein,the problem now remains that the protein must be further purified insome instances, and recovered in a form wherein its biological activitycan be utilized.

Since the protein has been precipitated in vivo under cytoplasmicconditions, it is clear that conventional solubilization techniques willfail. Accordingly, a more drastic means is required to bring thisprotein into solution so that it can be used. It has been found that astrong denaturing solution is effective in doing this. However, theresulting solution may or may not provide a biologically activepreparation.

In addressing the problem of utilizing this solution which contains bothstrong denaturant and solubilized refractile protein to recoverbiological activity as shown by appropriate assays, the most "obvious"alternatives fail to yield successful results: Dilution, if necessary,with larger amounts of the same "solvent"--i.e. more of the strongdenaturing solution, to obtain the proper concentration for biologicaltesting is clearly undesirable because the strong denaturant itselfwould interfere with biological activity. Dilution of the solution withdilute buffer or with water is also unworkable in that reprecipitationof the refractile protein almost invariably occurs; even if dilutiondoes not result in precipitation, the expected levels of activity areoften not shown.

There are a limited number of successful alternatives for recoveringbiologically active product which are consistent with the general schemeof purification herein described. One of these is replacing the strongdenaturant with a weaker one, followed by reduction of the concentrationof the weak denaturant. This procedure appears to preserve thesolubility of the refractile protein, and provide a medium which itselfdoes not interfere with the biological activity. While it has been foundthat in some cases, this procedure alone does not result in positivebiological activity, in other cases it does. The fusion protein formedwhich includes a foot and mouth disease (FMD) viral coat protein is onesuch instance. This also seems to be the case with respect to animalgrowth hormones. Where these circumstances occur, it is sufficient todissolve the pellet in the strong denaturant, buffer exchange into aweak denaturant, or weaken the chaotropism by limited dilution, furtherpurify it, if desired, using conventional techniques, and finally tobuffer exchange gradually into more dilute solution.

However, according to presently known data, success of this scheme inrecovering biologically active protein seems to be the exception ratherthan the rule. In many instances, apparently, more positive steps needto be taken to "renature" the protein which has been dissolved. Thedenaturation which has taken place may be the result either of originalmisfolding in the bacterium or of the isolation conditions or both. Inany case, it appears prudent to unfold and refold the resulting proteinusing one of the three following approaches, all of which require bufferexchange into weak denaturant (which is, alone, sufficient in somecases) prior to reformation of disulfide bonds. In one approach, theprotein is simply further purified under reducing conditions whichguarantee the conversion of any disulfide linkages to sulfhydryl groups,to permit it to refold itself under the conditions of purification, andthen to allow air or some other oxidizing agent to reform the disulfidebonds when the proteins have been properly refolded. A second approachis to break any disulfide linkages which may have been formedincorrectly, by sulfonating the protein, again allowing the protein torefold under more amenable conditions, and then reforming the disulfidebonds by removal of the sulfonate using a sulfydryl reagent in both itsreduced and oxidized (disulfide) form. A third alternative is to allowthe refolding to occur in the presence simply of the proper solutionenvironment and in the presence of a sulfohydryl-disulfide combinationso that sulfhydryl and disulfides are constantly being formed andreformed.

In any event, this "renaturation process" appears best to take placeconcommitant with further steps for purification of the protein i.e.ridding it of the minor contaminants that it contains after simpledissolution from the pellet.

This application, then, is directed to an invention which in its variousaspects, provides a successful protocol for heterologous proteinpreparation.

In one aspect, the invention concerns a process for isolating therefractile protein from background host cell protein through lysis of asuspension of host cells followed by recovery of the refractile bodiesthrough centrifugation at low speed. The course of this purificationprocess can conveniently be followed by examination of the preparationunder a microscope to determine the presence or absence of fragments ofbacterial cell walls, fragments or whole cells.

In another aspect, the invention concerns a process for enhancing theamount of heterologous protein which is precipitated in refractile formprior to isolation of the refractile bodies. The process involvestreating a suspension of the host cell culture with a killing protocol,such as high acid concentration, heat, or low concentrations of nonpolarorganic solvents. This "kill" treatment results in further precipitationof heterologous protein, and is then followed by the proceduresdisclosed in other aspects of the invention to recover the increasedamounts of precipitated protein.

In a third aspect, the invention concerns a process for recovering therefractile protein in a usable form by solubilizing it in a stronglydenaturing solution. This process may contain the additional step oftreating a suspension of the host cells in a buffer of suitable ionicstrength to solubilize the host cell protein, before recovery of thedesired refractile protein.

In a fourth aspect, the invention concerns a process for furtherpurification of the refractile protein previously solubilized in astrongly denaturing solution, preferably in the presence of a reducingagent, which comprises separation away of large molecular weightcontaminants using either a molecular sieve or high speedcentrifugation.

In still another aspect, the invention concerns steps to utilize orfurther purify the heterologous protein already dissolved in stronglydenaturing solution, wherein the solution is modified by weakening thedenaturing medium by dilution or by exchange with a weak denaturant,optionally in the presence of reducing agent. Under the influence ofweakly denaturing conditions, in some instances, aided by the reducingagent available, refolding is permitted to take place. The weakdenaturant replacement is also advantageous because it permits furtherpurification and/or renaturation steps to be conducted which would failin strongly denaturing surroundings.

Such replacement can conveniently be done by buffer exchange against acomparable concentration of a denaturant whose chaotropic properties areinherently weaker (such as, for example, replacing guanidine with urea)or by dilution (if the solubility characteristics of the subject proteinwill permit) to a decreased concentration of the same strong denaturant.

Still another aspect of the invention concerns a specific reactivatingprocess which is carried out in the presence of a weakly denaturingmedium. In this process, the refractile protein, which is solubilized ina strongly denaturing solution, is treated with a mild oxidizing agentin the presence of sulfite ion. This converts cysteine and cystinecontaining proteins to the protein-S-sulfonates. The strongly denaturingsolution is then weakened to permit refolding, and disulfide linkagesare reformed using a sulfhydryl compound such as, for example,β-mercaptoethanol or reduced glutathione, in the presence of thecorresponding disulfide (oxidized) form.

In another aspect, the protein solubilized in strong denaturant isfurther processed by refolding which comprises replacement of the strongdenaturant with a weak one and treating with a mixture of a sulfhydrylcompound, predominantly, along with its disulfide form in lesser amount,i.e. a one-step refolding procedure in "redox buffer".

In still another aspect the invention is directed to purificationprocedures carried out in the presence of a reducing agent such as, forexample, β-mercaptoethanol followed by the denaturant and reducing agentbeing removed by dialysis or other suitable means. If no pains are takento exclude air, this then serves to reoxidize the protein to reformdisulfide linkages, formation of which had been thwarted by the presenceof reducing agent.

The invention also relates to a "standard" multistep process forpurification of heterologous proteins precipitated in host cell cultureswhich comprises the steps of removing the soluble background hostproteins in a solution of proper salt concentration and pH, followed bysolubilizing the heterologous precipitated protein in a denaturingsolution containing a reducing agent, and recovering from the denaturingsolution the desired protein in renatured form. Additional steps toachieve further purification of the desired protein are optional and maybe selected from a number of conventional techniques but are carried outin the presence of reducing agent. These steps preferably comprise, forexample, size separation by gel permeation chromatography, and removalof undesired proteins by differential adsorbtion on an ion exchangeresin.

This aspect of the invention provides a general procedure which isapplicable to precipitated heterologous proteins in host cell culture,regardless of their biological nature and thus has the advantage ofoffering uniformity of equipment requirements for any desired product.This procedure is applicable generally with only minor modification oradjustments being required for specific proteins.

Finally, the various aspects of this invention by suitable combinationand selection thereof, provide a solution to the problem of refractilebody protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the cell paste, under a phase contrast microscope, of E.coli cells expressing a fusion protein containing urokinase (UK).

FIG. 2 shows a suspension of pellet resulting from low speedcentrifugation of the disrupted cells from FIG. 1 (again, as seenthrough a phase contrast microscope).

FIG. 3 shows the results of SDS PAGE on preparations of pGH, rabiesantigen, and urokinase, contrasting the content of impurities to thedesired protein in the crude sonicate of cells with that of isolatedrefractile bodies prepared according to the method of the invention.

FIG. 4A is a photograph of the partially isolated refractile bodies ofhuman growth hormone isolated from E. coli.

FIG. 4B is a photograph of an SDS PAGE gel contrasting the whole celllysate with the refractile body protein content for both live and killedcells.

FIG. 5A is a photograph of E. coli whole cells containing refractilebodies of pGH.

FIG. 5B is a photograph of the sonicate of the preparation in 5A.

FIG. 5C is a photograph of the centrifugation pellet when the sonicatedsuspension of B is centrifuged at low speed.

FIG. 6 shows the results of SDS PAGE on supernatant and pellet fractionsof killed and nonkilled cells.

DETAILED DESCRIPTION OF THE INVENTION

(A) Definitions:

"Heterologous" proteins are proteins which are normally either notproduced at all by the host cell, or normally are produced only inlimited amounts. The advent of recombinant DNA technology and otherstandard genetic manipulation such as point mutagenesis, has enabled theproduction of heterologous proteins in copious amounts from transfectedhost cell cultures. In practice, these heterologous proteins arefrequently produced by genetic expression in quantities that involvetheir precipitation under conditions which maintain the solubility ofhost cellular proteins.

In some instances, the insolubility of the expressed protein is suchthat these proteins are present in the host cell as so-called"refractile bodies," i.e., bodies which refract light and appear asbright spots when viewed through a phase contrast microscope. Hence, theproteins are often referred to as "refractile proteins" or "refractilebody proteins".

The invention herein is directed to procedures which are useful inisolating, purifying, and, if necessary, reactivating proteins whichappear in host cells in the form of "refractile bodies". Part of theinvention concerns methods which encourage such refractile bodyformation; however, the procedures for protein recovery and activationdisclosed herein are intended to be specifically applicable to suchrefractile proteins.

In the herein specification "refractile", "desired" and "heterologous"are used interchangeably to denote a protein expressed in a foreign hostwhich is, at some stage of expression or purification, visible by phasecontrast microscope as a precipitate, regardless of the physical stateof the protein at the time it is referenced. E.g. "refractile" proteinwill still be used in some instances to denote said protein even afterit has been converted from refractile to soluble form by the process ofthe invention.

Various heterologous proteins expressed in bacterial host cells, forexample, pGH, hGH, and viral coat proteins such as a fusion protein withFMD virus, protein and HBsAg form refractile bodies to a greater orlesser extent under commonly found culture conditions. Certain otherproteins such as immune interferon (IIF) and leukocyte interferon (LeIF)are more soluble in the cytoplasm. (Fibroblast interferon (FIF) is,however, refractile in host culture.)

"Host cells" includes, where used in the context of a starting materialin a procedure description for heterologous protein isolation, any ofthe forms in which the cells could be so used. It includes, for example,besides the harvested cell paste, the entire cell culture, a frozensample of the paste, or a frozen and thawed sample of the paste. Thusthe phrase "treating host cells in a buffered solution" may refer, forexample, to manipulation of the whole culture broth or to a preparationusing spun down cells.

"Reactivation", as used herein, is almost synonymous with"refolding"--i.e., it refers to assurance of biological activity for aprotein preparation by placing it in a conformationally active form."Reactivation" does not, as defined herein involve any change in theamino acid sequence and does not include, for example "activation" ofthe type wherein peptide precursors are cleaved to their active forms,such as conversion of trypsinogen to trypsin or prorennin to rennin.

"Biological activity" will refer to the activity of the protein in vivo,to its activity in conventional in vitro and in vivo biological assaysdesigned to test its functionality, to its ability to elicit an immuneresponse, or to the ability of the protein to react with antibodies tothe native protein. It is to be noted that in some cases, for example,proteins are "biologically active" when tested for reactivity with theappropriate antibodies, but not in functionality assays. However, asantibody reactivity is generally the most straightforward and easilyperformed assay method, it is sometimes used as a convenient measure of"activity".

"Ionic strength" refers to the conventional measure of ion concentrationin aqueous solution. It is defined as 1/2 of the sum (over all ions insolution) of the product of the concentration of each ion, times thesquare of the charge thereon.

"Denaturing solution" refers to a solution which contains a"denaturant". "Denaturant", as used herein, refers to those chaotropiccompounds or materials which, in aqueous solution and in suitableconcentrations are capable of changing the spatial configuration orconformation of proteins through alterations at the surface thereof,either through altering, for example, the state of hydration, thesolvent environment, or the solvent-surface interaction. Examples ofsuch denaturants include urea, guanidine hydrochloride, sodiumthiocyanate and detergents, such as SDS and Triton. It does not includesuch drastic and irreversible denaturing procedures as high temperatureand high acidity.

It will be noted that some of the listed reagents are strongdenaturants, while others are weaker, and that, of course, theconcentration of any of these will directly affect its strength andeffectiveness. There can be no specifically exact dividing line between"strong" and "weak", however, strong denaturing conditions morecompletely "unfold" the protein from whatever conformation it wouldspontaneously have preferred due to its amino acid sequence havingconferred areas of hydrophilicity and hydrophobicity along the chainunder physiological conditions. The most commonly used stronglydenaturing environment useful in dissolving refractile protein is afairly high (4-9M) concentration of the ionic denaturant, guanidinehydrochloride. Urea is the most frequently used example of a weakdenaturant as even fairly high (e.g. 7M) concentrations permit theretention of some protein secondary structures, and provide a route torefolding to the "native" conformation. It happens also to be nonionicin character, which is significant with respect to its use in thoseaspects of the invention which entail the use of, for example, ionexchange techniques.

Accordingly, a "strongly denaturing" solution refers to a solution whichwill effectively "unfold" a protein also dissolved in the solution. Theunfolding will be relatively extensive, but reversible. Solutes whichare effective in carrying out unfolding to this extent are exemplifiedby guanidine hydrochloride and sodium thiocyanate, usually in relativelyhigh concentrations in the range of approximately 4-9M, and detergentsusually supplied in concentrations of the order of 0.01-2 percent.

"Weakly denaturing solutions" refers to those solutions which permit atleast partial folding of a protein into the spatial conformation inwhich it finds itself when operating in its active form under endogenousor homologous physiological conditions, and also solubilizing anyintermediate forms between the "denatured" form as would be found in astrongly denaturing solution, and the properly folded conformation.Examples of such weakly denaturing solutions are high concentrations ofurea, ordinarily in the range of 4-9M and low concentrations of thedenaturants set forth above which, in high concentrations, are stronglydenaturing. These latter "low" concentrations are ordinarily in therange of 0.5 to approximately 2M. Occasionally, however, the functionalstatus of "weakly denaturing solution" can also be observed simply underfairly standard enzyme assay conditions such as, for example, low bufferconcentrations of the order of 0.1M and below, and physiological pH. Asused in this invention, "weakly denaturing solution" refers to thefunctional definition--i.e. those solutions which permit refolding fromwhatever contorted conformation the protein has, for whatever reason,assumed through intermediates soluble in this solution, to aconformation which is capable of exhibiting biological activity.

There are abbreviations and descriptions conventionally used with regardto particular techniques that are used in this invention, and forconvenience these will be described briefly here:

Gel permeation chromatography or gel filtration is a commonly usedpurification technique which discriminates between molecules accordingto their size. This is also frequently referred to as a "molecularsieve". By suitable selection of the gel, almost any size range can beselected for. Molecules which are large enough to be excluded from thegel pores are passed unretarded through a column containing the gel;smaller molecules are fractionated by the column.

SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) isa conventionally employed technique which permits determination ofapproximate molecular weight and purity. In this technique, the proteinpreparation is electrophoresed under reducing conditions in the presenceof detergent. The extent of migration for a particular molecule isdependent only upon molecular weight as determined in the absence ofdisulfide linkages (due to the reducing conditions). Therefore, thequantity of a particular protein present in a preparation can beestimated by densitometry measurements on a stained band appearing atthe position corresponding to the molecular weight of the protein. Adetailed description of this technique is found in Laemmli, U. K., etal. Nature, 227: 680 (1970) incorporated herein by reference.

"Western Blot" refers to an antibody specific binding technique whereina solution or suspension containing the protein to be measured isexposed to a nitrocellulose filter, which filter is then soaked with alabelled antiserum to the desired protein. The presence of the desiredprotein is ascertained by the retention of label on the filter due tothe insolubilization of the antibody by reaction with the specificprotein. A detailed description is provided by Towbin, H., et al., Proc.Nat. Acad. Sci. (USA.), 76: 4350 (1979) incorporated herein byreference.

"Chromatographic ion exchange protein purification techniques" refers toa series of procedures wherein material is subjected to chromatographicseparation based on an ionic exchange column interaction. Frequentlyused columns are, for example, DEAE cellulose, frequently denoted simplyas DEAE or, for example, DE-52 or DE-53 as common trade names, orcarboxymethyl cellulose (CMC). At appropriate pH values, a columncontaining DEAE behaves as an anion exchanger, and negatively chargedparticles bind to the column. Elution can be accomplished by from suchcolumns altering the components of eluting solvents, for example, byaltering the pH, the ionic strength, or dielectric constant of thesolution, or even through regulation of temperature.

"Buffer exchange" refers to techniques whereby the effective "solvent"i.e., the liquid environment of a macromolecule is changed. Thus, inthis sense, "solvent" really includes micromolecular solutes (e.g.salts) of the medium in which a desired macromolecule finds itselfsince, in fact, its solubility may be attributable to them. For example,in the process of a present invention, the desired protein may beprepared for ion exchange chromatography by providing a solventcomprising 8M urea in an appropriate buffer replacing, for example 7Mguanidine hydrochloride, which, in one preferred embodiment, is used asa denaturant. To make this "buffer exchange", one suitable technique isdialysis of the 7M guanidine hydrochloride solution containing theprotein against substantially larger quantities of the urea buffer.However, other buffer exchange techniques, such as, for example gelpermeation and diafiltration are also available and workable.

B. General Description

Scheme 1 depicted below shows the general procedures involved in solvingthe problem of isolating an active desired protein from host cellswherein this protein has been produced and deposited in the form ofrefractile bodies. ##STR1## C. Recovery of Refractile Bodies

As shown in scheme 1, because the refractile bodies are enclosed in thecells, it is desirable first to disrupt the cells so as to release therefractile bodies and make them available for recovery by, for example,centrifugation. In one aspect of the invention, purification ofrefractile proteins is obtained simply by insuring that the cellulardebris is sufficiently disrupted that it fails to appear in the pelletunder low speed centrifugation. In this aspect of the invention, thecells are suspended in a buffer at pH 5 to 9, preferably about 6 to 8,using an ionic strength of the order of 0.01M to 2M preferably 0.1-0.2M.Any suitable salt, including NaCl can be used to maintain a proper ionicstrength level. This ionic strength range is known to be adequate forthe invention, although the exact outer limits of permissible ionicstrengths are not clearly understood or known. It is, apparently,undesirable to use essentially zero ionic strength, however. The cells,while suspended in the foregoing buffer, are then lysed by techniquescommonly employed such as, for example, mechanical methods such as useof a Manton-Gaulin press, a French press, or a sonic oscillator, or bychemical or enzymatic methods such as treatment with lysozyme.

When it is judged that the cells are sufficiently disrupted that therewill be a minimum of, or no, cellular fragments of sufficient size to bespun down remaining, the suspension is centrifuged at low speed, around500 to 5000 times gravity, preferably around 1000 times gravity in astandard centrifuge for a suitable time period depending on volume,usually about 10 minutes to 1/2 hour. The resulting pellet containssubstantially all of the refractile proteins, but if the cell disruptionprocess is not complete, it may also contain broken cell fragments.Completeness can be assayed by resuspending the pellet in a small amountof the same buffer solution, and examining the suspension with a phasecontrast microscope. The presence of broken cell fragments indicatesthat further sonication or other means of disruption is necessary inorder to remove the proteins associated with these fragments. After suchfurther disruption, if required, the suspension is again centrifuged,and the pellet recovered, resuspended, and reexamined. This process isrepeated until visual examination shows the absence of nonrefractileproteins in the pelleted material. With suitable preparations, theconditions for particular protein may be defined sufficiently clearlythat in carrying out the process of the invention, only one suchsuspension, disruption, and centrifugation is required. However, even inthis case it is preferable to carry out the process in the above severalsteps, most preferably a total of three as this permits a desirablereduction in required volumes of aqueous buffer (i.e. the amount used toresuspend the pellet is substantially smaller than the volume used inthe original preparation), and the quality of the preparation isconsistently assured by visual monitoring.

The proteins in the pellet so prepared contain from about 40 percent toover 90 percent of the desired heterologous protein as compared to totalprotein contained therein, depending on the specific protein produced bythe host cell.

For example, when prepared by the particular procedures utilized inExamples 1 through 8, for human growth hormone more than 90 percent ofthe refractile protein was in fact the desired protein (as measured bythe methods there indicated), while only about 50 percent was thedesired protein in the cases of human interferons and of the viralantigenic proteins. Intermediate amounts were obtained of tissueplasminogen activator and calf rennin. These purities are, at thisstage, adequate for some uses of the desired protein.

The pellet is capable of being dissolved in a solution of denaturant,which resulting solution as such may or may not contain an active formof the protein. The results of utilizing this method alone for isolatingrefractile proteins is exemplified in the aforementioned Examples 1through 8 of the specification. It is most advantageously applied toheterologous proteins produced in bacterial culture, most preferably E.coli and for the isolation of proteins selected from the groupconsisting of hGH, bGH, pGH, human fibroblast interferon (FIF), humanimmune interferon (IIF), human tissue plasminogen activator (tPA), calfprorennin, and FMD coat proteins. The technique of dissolving thepelleted proteins in a denaturant and of recovering activity areborrowed from the other aspects of the invention, which aspects are setforth in the succeeding paragraphs.

D. Enhancing Production of Refractile Bodies

In a second aspect, the invention is related to a procedure forenhancing the quantity of protein, expressed in a foreign host cellwhich is insolubilized in refractile form, prior to purificationthereof. In the case of many proteins, when growth and expression areobtained as set forth in the examples herein, enhancement is unnecessarybecause virtually all of the desired protein appears in the form ofthese refractile bodies, anyway. Exemplary of such proteins are animalgrowth hormones, and fibroblast interferon. However, in the case ofhuman growth hormone, apparently only roughly 50 percent of the proteinexpressed by E. coli appears as refractile bodies, and a correspondingloss in yield will be experienced if these procedures are not followed.Similarly, immune interferon is largely produced in nonrefractile form,and if procedures directed to refractile bodies are to be employed forits isolation, enhancement must be effected by the methods set forthwithin this aspect of the invention.

In this procedure, advantage is taken of the desirability of utilizing a"kill" step whereby the recombinant cells can be brought intoconformance with government regulations directed toward safety in thegrowth and harvest of such cells. A number of killing techniques areavailable, and have been carried out simply for safety purposes, butthey have the additional desirable effect in the aforementionedinstances of increasing the quantity of refractile protein. In theprocess of this aspect of the invention, the host cells may be killedeither in the medium in which they are being cultured and grown, or in asuspension prepared by an initial centrifugation or other methods ofconcentration of the cells of the original medium, recovery of a cellpaste, and resuspension in an aqueous solution. Suitable kill proceduresare administration of low concentrations of acid, heat treatment, and,most preferably, treatment with nonpolar organic solvents in smallpercentage.

In a particularly preferred procedure, the culture medium is brought to0.25 percent in phenol and 0.25 percent in toluene and allowed toincubate at room temperature to 45° C., preferably about 37° for 15minutes to several hours most preferably 0.5 hours. Alternatively, iffacilities are available for containment, the cells are first harvestedunder containment, resuspended in for example 0.01M-2M ionic strength,preferably 0.1-0.2M ionic strength buffer of pH 5-9, preferably 6-8. Thesuspension is then treated with low concentrations of organicsolvents--e.g. 0.25 percent each in phenol and toluene.

In other embodiments, either the cell medium or suspension as abovedescribed can also be heated to about 60°-80° C. for about 15 min.-45min. to effect killing; or brought e.g. to a pH of about 0.5-1.5.

These procedures result in considerable additional percipitation of theexpressed heterologous protein, if it is not already so percipitated.Example 9 sets forth a particular instance in which this method isadvantageous.

The procedure of this aspect of the invention can then be combined withfurther techniques, disclosed herein, for the recovery of activeheterologous protein.

E. Solution of Heterologous Protein in Strongly Denaturing Solution

In a third aspect, the invention concerns a procedure for dissolving therefractile proteins from their insoluble or pelleted form by using astrong denaturing solution. While the proteins in refractile bodies aregenerally insoluble under the conditions prevailing in the cytoplasm(and thus in relatively weak ionic strength buffers) they appear to besoluble in fairly high concentrations, typically 4 to 9M concentrations,of certain denaturants. In the process of the invention, strong, oftenionic, denaturants are apparently the most practical. A particularlypreferred denaturant is a guanidine salt, although detergents such asTriton and SDS and salts of thiocyanate ion have also been usedsuccessfully. A range of 4 to 9M concentration is workable for guanidinesalts or sodium thiocyanate, with 6-8M, being particularly preferred.Detergents are used in the range of 0.01-2 percent of solution. The pHof the solution must be compatible with the characteristics of theparticular protein, so that irreversible denaturation or proteinhydrolysis does not occur; optimum concentration of denaturant isdependent on the protein to be solubilized and the pH used.

While solubility, once achieved is often maintained when the solubilizedheterologous protein is exchanged into a more weakly denaturing medium,initial solubilization in this same weakly denaturing medium is notpractical. Whether for thermodynamic or kinetic reasons, the proteindoes not dissolve within a reasonable time under these less drasticconditions.

Additional components may be added to the solution to maintain thedesired pH level, as may other ancillary components desirable inparticular instances such as, for example, chelators such as EDTA. Asshown by the behavior of the proteins in refractile bodies in Examples1-8, 10, and 11, solutions which are not strongly denaturing typicallyfail to dissolve these refractile proteins (although host cellularproteins are dissolved), while strong denaturing solutions do dissolvethem. Accordingly, such weakly denaturing buffers can also be used tosolubilize and remove host cell proteins.

In the process of this aspect of the invention, the host cells are firstsuspended in a medium of correct ionic strength to solubilize many hostcell proteins--i.e. 0.01-2M ionic strength, preferably about 0.4-0.6Mionic strength at a pH of about 5-9, preferably around 6-8. Precise pHand ionic strength limits cannot, or course, be set forth, but workableranges are here given.

The cells are disrupted in the presence of the forgoing solution, andthe suspension centrifuged to form a pellet. The pellet contains mainlythe desired protein in refractile form, and it remains to employ thestrongly denaturing medium described above to solubilize it. Thesolubilized protein may then be recovered using means which allow itsrenaturation.

F. Removal of High Molecular Weight Contaminants

In a fourth aspect, the invention is directed to a process for freeingthe solubilized, desired, previously refractile protein from highermolecular weight components directly from the strongly denaturingsolution, even if the denaturant is ionic, using either a molecularsieve or high speed centrifugation. The procedure follows the left-mostseries of arrows from the "supernatant containing desired protein" inScheme 1, wherein the supernatant from the pellet which has beenextracted with strong denaturant is either passed over a column of asize-discriminating gel permeation molecular sieve, such as sephacryl,or is centrifuged at high speed to bring down higher molecular weightcomponents. Neither of these separation procedures requires removal ofions from solution, and hence can be carried out directly on the extractfrom the pellet, even if this extract is ionic. (See Examples 10 and11.)

In carrying out the removal of high molecular weight impurities throughgel filtration, a column containing a molecular sieve, such as, forexample, Sephacryl S-300 is equilibrated in a suitable buffer(containing, preferably, a reducing agent) and the solution containingthe heterologous protein passed through the column. The high molecularweight flow-through volume is descarded, and the heterologous proteinthen eluted with further amounts of buffer. Eluted protein may bemonitored for example by measurement of optical density at 280 nm, andthe presence of the desired protein verified by dialysis against anon-ionic solvent, followed by SDS-PAGE to ascertain the correctmolecular weight protein.

In the alternative approach, high speed centrifugation is carried out byspinning the protein at 25,000-40,000 xg, preferably 35,000 xg for 10min.-3 hrs, and recovering the supernatant for further purification.

Use of gel permeation chromatography as a first chromatographic step ina commercial purification process for protein, i.e. carrying out gelpermiation prior to, for example, ion exchange chromatography, isunusual. However, in the process of the present invention the proteinhas a high level purity (virtually always as high as 50 percent or more)after just the lysis and/or denaturant extraction steps. Therefore, ascompared to conventional procedures for the isolation of proteins, thedesired protein is in a fairly high state of purity before it issubjected to the gel filtration step. Thus, the usualdisadvantage--i.e., the lower capacity of gel filtration as compared toion exchanges does not pertain in this case. Since the amount ofimpurities is small, a total high capacity is not needed as it would beto isolate a small amount of a particular protein from a largecollection of impurities.

Further purification may optionally be carried out consistent with thisaspect. If the dissolving denaturant was ionic, desalting the solutionby exchanging into non-ionic denaturant is required if such furthersteps involve ion exchange. As a practical matter, it is preferable toutilize a weakly denaturing solution such as urea. While in principlethe denaturant might simply have been removed by, for example, dialysisinto standard types of buffer, this often results in reprecipitation ofthe protein. Maintaining the protein in a solution which still containsa reasonable denaturant concentration prevents premature precipitationof the protein. Once the ions have been removed and replaced by anon-ionic substance, a variety of chromatographic techniques involvingion exchange or neutral adsorption supports may be used for furtherpurification. An advantageous choice among these is DEAE cellulosechromatography at such a pH that the desired protein fails to stick tothe column and appears in the flow through volume. The column thuscaptures the anionic protein contaminants, and removes them from thedesired protein. This approach, rather than the converse, wherein thedesired protein is adsorbed and eluted, has the clear advantage ofsimplicity and of more limited resin requirements. Since the desiredprotein predominates in quantity, only sufficient resin to adsorb thecontaminants is required. However, the process addressed by this aspectof the invention is not limited by any specific example, but ratherpermits the use of a variety of separation techniques as furtherpurification methods.

G. Maintaining Solubility Without Strong Denaturant

Still another aspect of the present invention comprises the maintenanceof solubility during purification by replacing the strongly denaturingsolution of the subject protein with a weakly denaturing solution priorto subsequent purification or biological testing. In some cases, forexample that of hGH expressed in E. coli, this alone may be sufficientto effect refolding, but this is not always the case. Further, in someinstances and for some applications (e.g. where subsequent ion exchangeis not involved) limited dilution of the strongly denaturing solutionwill suffice to maintain solubility for some proteins. However, a bufferexchange procedure whereby the strongly denaturing solution is replacedby a weaker one, is useful to maintain solubility while permittingfurther purification, and in some cases, restoration of biologicalactivity. This is desirable, in particular, in the case of ionic strongdenaturants, because, for example, it is often necessary to utilize ionexchange techniques in order further to purify partially purifiedrefractile proteins. It is not possible to utilize the solution directlyresulting from solubilization of refractile proteins because the ionicdenaturant interferes with ion exchange. However, removal of thisdenaturant altogether often results in precipitation of the desiredprotein. These problems can be avoided by buffer exchanging an ionicstrong denaturant such as guanidine, with a less powerful non-ionic onesuch as urea. In particular, urea appears, in reasonableconcentrations--i.e. 1-9M approximately, both to permit refolding of theproteins into something approximating their native state whethersupplied in the form extracted, or in S-sulfonated form as set forth inanother aspect of this invention, and also (perhaps because of this)maintain solubilization.

The "buffer exchange" may be done in the presence of β-mercaptoethanolor another suitable reducing agent so as to maintain reduction of anyimproper disulfide linkage which might have been formed prior to thebuffer exchange renaturing process or, alternatively, with the proteinin the form of a S-sulfonate.

Accordingly, in the process of this aspect of the invention, thestrongly denaturing solution containing the subject protein or itsS-sulfonate and, for example, 4-9M guanidine HCl is buffer-exchangedusing dialysis or diafiltration against a solution of urea or other weakdenaturant which optionally contains a suitable concentration ofreducing agent, before any subsequent purification takes place. (As setforth in another aspect of the invention, the original stronglydenaturing solution may first treated with sulfite and a mild oxidizingagent in order to conduct sulfitolysis prior to buffer exchange againsta weakly denaturing medium. This sulfitolysis procedure is notinconsistent with the scope of the present aspect of the invention.) Ineither event, the weakly denaturing solution contains protein which isfolded more nearly to the form corresponding to the biologically activeprotein, (whether S-sulfonated or not) and the resulting solution can besubjected to the full panoply of purification techniques, such as ionexchange on an anion column such as DEAE cellulose or on a cation columnsuch as CMC. In any case, the subsequent purification methods areconducted in a conventional manner at appropriate pH's and saltconcentrations depending on the particular protein to be isolated, andon the specific strategy to be employed. Such subsequent purificationmethods are well known in the art and their application is familiar tothe practitioners thereof.

H. Refolding

The remaining three aspects of the invention represent alternativeprocedures directed to reactivating an inactive (presumably because itis incorrectly folded) form of the desired protein.

In the first such aspect, refractile proteins which have beensolubilized in a strong denaturant such as guanidine hydrochloride arerenatured through preliminary sulfitolysis in the strongly denaturingsolution followed by refolding, sulfonate deletion, and disulfideformation, in a weakly denaturing medium in the presence of a sulfhydrylcompound containing a small percentage of its corresponding disulfideform. The disulfide form may either be supplied directly, or thesulfhydryl compound used alone in the absence of precautions to excludeair. This creates a suitably oxidizing atmosphere sufficient to insurethe presence of some disulfide.

Typically, to carry out the sulfitolysis, the solubilized refractileprotein in a strongly denaturing medium, such as 4-9M guanidinehydrochloride is brought to approximately 5-200 mg per ml, preferablyaround 15-30 mg per ml in sodium sulfite, or corresponding molar amountsof other sulfite salts, in the presence of a mild oxidizing agentsufficient to regenerate disulfide from any sulfhydryl groups whichresult from the reaction. Suitable oxidizing agents are, for example,molecular oxygen with catalysis by metal cations or sodiumtetrathionate, preferably sodium tetrathionate. Sodium tetrathionate isadded in the amount of approximately 1-20 mg/ml preferably about 10 mgper ml corresponding molar amounts of other agents may be used. Thesolution is then allowed to stand 4-24 hours, preferably overnight, at15° C. to 35° C. preferably around room temperature. While suitableranges of concentrations and temperatures, etc. have been given, theprecise conditions which are most advantageous depend, of course, on thenature of the protein to be sulfitolyzed.

Furthermore, only "partial" sulfitolysis is sometimes useful. In thatinstance, much lesser amounts of the sulfite and oxidizer may be used.See, e.g., Example 13. The foregoing amounts are merely workableguidelines and the outer limits are defined by various parametersincluding the amount of protein in solution and the completeness ofsulfitolysis desired.

In the above sulfitolysis reaction, the disulfide bonds are broken and asulfonate substituted for one of the sulfide partners. It is believedthat the mechanism of this reaction involves a nucleophilic attack bythe sulfite ion to break the disulfide bond. In any event the resultinglinkage is protein-S-SO₃, i.e., a protein-S-sulfonate.

The resulting protein S-sulfonate solution is then placed into a weaklydenaturing solution either by dilution or by buffer exchange, e.g. bydialysis into a solution containing a weak denaturant such as urea.

It is to be noted that further purification using ion exchangechromatography or other standard protein purification techniques may beused while the protein is still in the S-sulfonated form.

The weakly denaturing medium provides a route to proper refolding, theprotein no longer being trapped by incorrect disulfide linkages. If ureais used as the weakly denaturing solution, appropriate concentrationranges are 1-9M, preferably 6-8M. The pH is kept at approximately 5-9,preferably around 6-8 with suitable buffer, and optionally with addedEDTA or other chelating agent. If dilution is used, appropriateconcentrations are about 0.5M-2M in the original strong denaturant. Tothe weakly denaturing medium, a system containing a sulfhydryl compound(RSH) and its corresponding disulfide (RSSR), for example,β-mercaptoethanol, reduced glutathione, cysteamine, or cysteine andtheir corresponding oxidized forms, preferably glutathione in thereduced (GSH) and the oxidized (GSSG) forms, is added. The pH isadjusted to a value such that the sulfhydryl compound (RSH) is at leastpartially in ionized form (RS⁻) so that nucleophilic displacement of thesulfonate is enhanced. Alternatively, the reduced form alone in thepresence of air may be used, as sufficient disulfide will be generatedin this environment. Typically the RSH to RSSR molar ratio isapproximately between 20:1 and 5:1, preferably about 10:1 and the totalglutathione or other reagent concentration in the 0.05 to 5 mM range.The mixture is incubated at about 0° C. to 37° C., depending on theprotein, 4-24 hours, preferably overnight.

While the sulfhydryl compound itself would be sufficient to effect theconversion of the protein S-sulfonate to the corresponding disulfide, orat least to form disulfide linkages with the sulfhydryl compound itself,the presence of an oxidized form is required to insure that suitabledisulfide linkages will remain intact. If unadulterated sulfhydrylcompound is added under conditions wherein oxidation is not permitted,the protein will ultimately wind up in the sulfhydryl form, rather thanas a disulfide. In order to prevent this, the oxidation potential of thesurrounding buffer is maintained by supplying a small amount of thedisulfide either directly or by permitting air oxidation of the reducedsulfhydryl.

The resulting solution now containing properly refolded subject protein,which presumably is secured by the correct disulfide linkages, may thenoptionally be stripped of denaturant by dialysis against suitable buffersolution of pH 5-9, and optionally containing small amounts of reducedglutathione or other sulfhydryl compound of the order of approximately 1mM in concentration. If the subsequent uses of the protein are feasiblein the presence of the denaturant, however, this step is unnecessary.

In foregoing procedure, the protein concentration is kept at a fairlylow level, preferably less than 1 mg per ml because in some cases(though not in all), higher concentrations are detrimental to theprogress of the reaction.

Further, the sulfitolysis reaction may optionally be carried out in ureaor other weak denaturant as well as in the strongly denaturing solution,and it may even be advantageous to do so, particularly in instanceswhere the denaturant concentration used to effect solution isparticularly high. In such cases, the buffer exchange into weakerdenaturant or dilution would be carried out before, rather than after,the sulfitolysis reaction.

In an alternative aspect of the invention designed to "refold" theheterologous protein, unfolding and refolding are made to take place inthe same solution by placing the subject protein or peptide into asulfhydryl/disulfide-containing buffer, which buffer has sufficientdenaturing power that all of the intermediate conformations remainsoluble in the course of the unfolding and refolding. A suitable mediumis for example 1-9M urea, preferably approximately 7M urea which appearsto be a weak enough denaturing agent that a close approximation to thecorrect conformation is permitted, and strong enough that mobility ofthe refolding chain, and solubility of the intermediates are possible.This embodiment may be characterized as "refolding in redox buffer."Both reduced (RSH) and oxidized (RSSR) forms of sulfhydryl compounds,for example, β-mercaptoethanol, glutathione, cysteamine, or cysteine,preferably glutathione are present in the appropriate exchange medium.

In this redox buffer refolding, the molar ratio of RSH to RSSR isapproximately between 20:1 and 5:1, preferably about 10:1, and the totalreagent concentration in the 0.05 to 5 mM range. The pH, again, must besufficiently high to assure at least partial ionization of RSH, althoughnot so high as to denature the protein. The mixture is incubated at 0°C. to 37° C., preferably about 5° C. for about 4-24 hours, preferablyovernight. Here, as above, the presence of both reduced and oxidizedforms of the sulfhydryl compound can be provided either directly, orthrough air oxidation of the sulfhydryl. Both forms need to be presentin order to maintain the proper oxidizing potential so as to precludecomplete reduction of the subject protein.

As is the case with other refolding processes of the invention, theprotein may, while in solution, be subjected to standard techniquesdirected to purification of protein such as gel filtration or ionexchange. It is particularly preferable to employ an ion exchangetechnique, such as DEAE cellulose.

In a still another aspect of this invention, refolding and restorationto a native form in the context of the purification process is done byretaining the protein in a reduced form throughout whatever purificationsteps are conducted, and reoxidizing in the presence of air to form theappropriate disulfide linkages upon final removal of the denaturant. Inthis process, a reducing agent is supplied in the initial solution ofrefractile protein in a strong denaturant and during all of thepurification steps subsequent thereto. Suitable reducing agents, are,for example, β-mercaptoethanol, dithiothreitol, and reduced glutathione,preferably β-mercaptoethanol. Upon removal of most or all of thedenaturant at the end of the process, the reducing agent is not includedin the reaction mixture, and sufficient air is present to reoxidize thesulfhydryl groups in the now properly folded protein to disulfidelinkages so as to secure the proper native form. Employment of such aprocess is exemplified in Example 10 and 11 herein.

H. A Standard Multistep Procedure

An abbreviated version showing the steps required in a general multistepprocess for purification which forms another aspect of the invention, aswell as two optional steps, selected from among those now conventionallyemployed, is shown as Scheme 2 below: ##STR2##

As shown in Scheme 2, the process in its basic aspect comprisesdispersing a cell paste in a buffer having an ionic strength ofapproximately 0.05-2.0M, preferably 0.4-0.6M, to complete or maintainprecipitation of the heterologous protein, and to dissolve or maintainthe solubility of the majority of the host proteins. After separationfrom host protein, the heterologous protein is dissolved in stronglydenaturing solution containing reducing agent. The process is completeupon recovery of the heterologous protein in biologically active formthrough buffer exchange. The gel filtration and ion exchange steps(Steps 3 and 4) are optional preferred embodiments of additionalpurification steps which may be appropriate in individual cases.

The cellular material used as starting material can be the whole cultureor a reduced form thereof such as cell paste. Bacterial cultures arepreferred, in particular, E. coli as host cells. Currently, it ispreferable to use a cell paste made subsequent to a killing step whichis employed to comply with current regulations promulagated as safetyprecautions. (The invention is applicable for protein recovery from hostcells whether or not a kill step is preliminarily employed.) In apreferred procedure, the culture broth, which is grown to approximately30-50 OD units at 550 nm is made approximately 0.25 percent each inphenol and toluene and allowed to stand for approximately 1/2 hour. Thisis successful in killing the cells without undue denaturation of thecellular protein. Heat and acid kills are effective but less preferred;however, these and other killing methods may be used, consistent withthe subsequent process of the invention. The killed materials thensubjected to the procedure of the invention may be either whole broth orthe centrifuged cells; however, it is preferable from a practicalstandpoint (of minimizing volumes) to use the centrifuged cell paste.This culture or paste may also be frozen for storage prior to thepurification process purely for convenience.

In conducting the initial extraction (Step 1 of Scheme 2) the cells aredispersed thoroughly in a solution which is buffered at approximately pH4-10, preferable from about pH 6-9, and contains ionic species at alevel of approximately 0.05-2.0M ionic strength, preferably about0.4-0.6M. Any appropriate buffer system may be used. The ionic strengthis provided by any salt, including the species used for buffering, but,for reasons of economy, preferably by sodium chloride. In addition, itis desirable that the buffer contain a chelating agent, such as, forexample, EDTA, and a reducing agent, for example, 2-mercaptoethanol(BME).

In carrying out the preceding step, it is highly desirable to obtain auniform suspension; if cell paste is used, mechanical dispersal ispreferred. There are dispersing machines available on the market forthis purpose, and a preferable choice is Dispax (Tekmar Inc.) Model SD45. However, if the broth culture is used, mechanical dispersal isunnecessary.

Since the heterologous precipitated (refractile) protein is usuallycontained within the cells, it is also necessary to homogenize the cellsuspension resulting from Step 1 using a homogenizer or press of a typethat will in fact destroy the integrity of the cells. A number ofdevices or techniques may be used, such as a French press, or a beadmill, or sonication; a Manton-Gaulin type 15M homogenizer is, however,preferred. When the cells have been dispersed and homogenized in thebuffer as described, the insoluble material is separated from thesoluble proteins preferably by centrifugation and the supertanant isremoved. The supernatant contains primarily the host proteins and isdiscarded.

In a preferred procedure, the pellet is washed by redispersing thepellet in a similar buffer in order to remove still further the hostmaterials from the insoluble proteins in the pellet. The washing iscarried out in the standard fashion by treating the pellet with a freshsample of the same buffer, redispersing and spinning down the washedpellet.

The pellet is then extracted as shown in step 2 to recover the desiredheterologous proteins. The pellet is dispersed in a strongly denaturingsolution by treating in a manner similar to that described in Step 1.Preferably, the solution used in this step would be 1-9 molar, mostpreferably 6-8 molar in a strong denaturant such as a guanidine salt,along with sufficient phosphate or other suitable buffering agents toprovide a pH of approximately 4-10. preferably about 6-9, and mostpreferably about 7, and preferably with small amounts of chelating agentsuch as, for example, EDTA. It is required that a reducing agent, suchas β-mercaptoethanol be present to insure conversion to free sulfhydrylgroups from any disulfide linkages. Other denaturants, of course, may beused. The pellet, when dispersed, is stirred with the denaturingsolution for up to 24-hours, preferably overnight.

The suspension is then spun down and the pellet, which containsundissolved and precipitated host protein and debris is discarded.

The purification to this point is sometimes sufficient that theresulting protein can be used after merely weakening the effect ofdenaturant through dilution or buffer exchange. If so, steps 3 and 4 canbe omitted, and step 5 performed directly as described below.

In step 5, the desired protein is then recovered in biologically activeform by replacing the denaturing agent with a suitable solvent medium.For some proteins, dilution to a lower concentration of the originaldenaturant will suffice. For others, buffer exchange into a differentdenaturant which is less chaotropic, such as urea is required. The finalstep in recovery is done in the absence of reducing agent to permitresecuring of disulfide bonds when the protein is allowed to unfold inthe weaker denaturant.

However, if further purification is desired, subsequent, optional stepsmay be taken in the presence of reducing agent to increase purity beforerenaturation. A number of choices of such steps may be made. Exemplaryand preferable among these are as follows:

In a first such preferred step, (step 3) the solution still containingthe denaturant reducing agent and the desired protein is chromatographedby a gel filtration process for size separation. The choice of theappropriate gel pore size, because of this size dependence, depends onthe nature of the protein to be purified. For the Foot and Mouth Disease(FMD) proteins the appropriate choice is, for example, Sephacryl S-300(Pharmacia).

The gel filtration step can be carried out in the presence of the highconcentration of an ionic denaturing agent which may have been used tosolubilize the desired protein. However, it is clear that an ionexchange chromatography step such as that exemplified in step 4, cannot.Therefore, if still further purification is desired based on ionexchange, the denaturing solution itself, if it contains ions, e.g.guanidine or even the eluate from the gel permeation chromatography ifit still contains these must first be subjected to ion removal. This canbe carried out by dialysis against preferably alkaline buffer againcontaining reducing agent with a high concentration of a neutraldenaturing agent such as, for example, approximately 8 molar urea, inorder to maintain the solubilization of the heterologous protein.

When the desalting has been accomplished, the material which is in thedialysis retentate is subjected to chromatography on an appropriate ionexchange column, such as, for example, DEAE cellulose. The conditionspreferably are selected so as to permit the desired protein to flowthrough the column in the void volume. This is advantageous, since lession exchange resin is required if it is the trace impurities that areremoved by absorption onto the resin, rather than the bulk of theprotein, which at this stage of the purification procedure is thedesired product.

The flow-through volume containing purified protein and (neutral)denaturant may either be used as such, in appropriate cases or freed ofthe denaturing agent by dialysis against a more dilute solution. It hasbeen found that in some cases a preliminary buffer exchange into a lowerconcentration of urea preceding a final buffer exchange into water orbuffer is required in order to prevent that precipitation of theprotein. Through all steps, until the last, reducing agent must bepresent. Suitable reducing agents are set forth, and the rationale fortheir use given, hereinabove in connection with the previously describedaspect of the invention.

The resulting solution is ordinarily of the order of 95-99 percent purewith respect to the desired protein. Recovery is typically of the orderof at least 50 percent and up to 98 percent of the heterologous protein.

The foregoing procedure may be applied especially advantageously to hGH,pGH, bGH, bovine interferon, tPA and FMD viral coat proteins.

I. EXAMPLES

The following examples are intended to illustrate the invention but notto limit its scope.

Examples 1-8 relate to that aspect of the invention which comprisessolubilization of the host cell proteins and recovery of the refractilebodies, as such, through low speed centrifugation.

Example 9 illustrates the enhancement of refractile body recoverythrough the use of a "kill" step.

Examples 10 and 11 relate to that aspect of the invention whichcomprises a multistep procedure combining a preliminary lysis andremoval of bacterial proteins, with solubilization of the resultingrefractile proteins and, inevitably, certain contaminants, in a strongdenaturant, followed by an optional subsequent purification regime whichhas, as a primary step, gel filtration or high speed centrifugation andrecovery of active protein. These Examples also illustrate those aspectsof the invention which the use of air as the disulfide forming reagentafter the protein has been allowed to refold in the presence of reducingagent, the ability of solvents which are strongly denaturing solutionsto dissolve refractile bodies, and the maintenance of solubility byexchange into weaker denaturant.

Examples 12, 13, and 15 set forth the refolding of at least partiallyinactive protein by sulfitolysis followed by treatment with redoxbuffer; Example 14 sets forth the "redox buffer refolding" process.

Example 16 specifically illustrates the efficacy of solution intostrongly denaturing solution followed by buffer exchange into a weakerdenaturant.

All references cited in these examples are incorporated into thisapplication by reference.

All of the examples relate to specific heterologous proteins which havebeen purified by the process of the invention. The details of thepurification will vary, of course, with specific proteins used. Althoughthe procedure of the invention will be similar in all cases, certaindetails, such as, for example, the selection of the denaturing agent insolubilizing the desired protein, the selection of appropriate sizinggels or ion exchange resins, as well as the ionic strength and pHconditions appropriate in each step, will be dependent on the nature ofthe protein. However, refractile proteins share enough properties incommon that these minor alterations will suffice to adapt the procedureto a particular protein in question.

EXAMPLE 1 Procedure for Production and Isolation of HeterologousProteins

A. Growth of Cells:

E. coli K12 cells transformed with recombinant plasmid pBR 322 carryingheterologous genes under E. coli trp promoter-operator control, weregrown in broth containing 10 g/l yeast extracts and 5 g/l tryptone to acell density of about 2-4×10⁸ cells/ml. 3-5 percent of the volume ofthis culture was inoculated into M9 medium (J. H. Miller, Experiments inMolecular Genetics, p. 431, Cold Spring Harbor Laboratory, 1972) orsimilar mineral salts medium containing 40-120 mg/l tryptophan. Thecultures were grown in a bench fermenter with sufficient agitation andaeration to achieve a growth rate of 60-90 minutes per cell division;glucose was fed to the cultures to maintain growth, but did not exceed50 g/l during the fermentation, and the pH of the cultures wascontrolled at 6.8-7.2 by NaOH or NH₄ OH. At cell density of 5-10 g dryweight/l, indole acrylic acid (IAA) or indole propionic acid (IPA) wasadded to the cultures to a concentration of 25-50 mg/l. Two to fivehours after the addition of IAA or IPA, the E. coli cells becameelongated and one or more refractile bodies per cell can be seen underphase contrast microscope at 1000-fold magnification.

B. Isolation of the Heterologous Protein:

The cultures were harvested by continuous centrifugation and the cellpastes were frozen at -10° to -20° C. (The cells may optionally bekilled before harvest by addition of 0.25 percent phenol and 0.25percent toluene added to the medium and incubating for 0.5 hrs at 37° C.(See Example 9).) Freshly harvested or frozen cell pastes wereresuspended in a buffer containing 10 mM Tris, 1 mM EDTA, pH 7.4 at aratio of 1 g cell paste to 10-40 ml buffer, and the cells disrupted bysonication or homogenization under high pressure.

Under phase contrast microscope, refractile particles were seen amongthe cell debris. FIG. 1 shows the suspension for E. coli K12 strain 3110(ATCC 27325) transformed with plasmid pUK 33 trp LE₂, described in U.S.application Ser. No. 368,773, filed Apr. 15, 1982 (abandoned in favor ofcontinuation-in-part Ser. No. 474,930, filed Mar. 14, 1983), expressinga fusion protein containing urokinase (UK). The refractile bodies appearas bright spots within the cell envelope. The suspension was subjectedto centrifugation at 1,000 x g (Sorvall SS-34 at 3,000 rpm) for 3-10minutes. After centrifugation, the supernatant was discarded, and thepellet was resuspended in the same buffer in 1/5 of the original volume.The suspension was examined under phase contrast microscope, and ifresidual intact cells or visible cell fragments were present, the aboveprocess repeated until visual examination of the resuspended pelletshowed only refractile particles. FIG. 2 shows the resuspended pelletfor the UK protein of FIG. 1. It appears that the preparation is mostlyrefractile with some cells and cell fragments included. If cells or cellfragments were present, suspension was subjected to disruption again.Isolation of refractile particles typically occurred after 3-4 cycles.The refractile particle preparation can then be stored frozen as pelletor in suspension, and is as much as 95 percent refractile protein.

To verify identity, the refractile particle preparations were subjectedto SDS-PAGE, Western blot, and/or radioimmunoassay (RIA). FIG. 3summarizes the purification results obtainable for pGH, rabies andurokinase. Rabies and pGH appear to result in single bands of protein inthe pellet, urokinase is more complex because of its several allotropicforms.

EXAMPLE 2 Human Growth Hormone

Recombinant DNA E. coli K12 cells carrying human growth hormone gene(strain W3110/p107) as described in U.S. Pat. No. 4,342,832 were grownin fermenter and harvested, and refractile particles isolated accordingto the procedure described in Example 1.

The particles showed a protein band corresponding to a molecular weightstandard of 22,000 daltons on 2-mercaptoethanol SDS-PAGE. A densitometerscan of the gel showed the amount of this protein was over 90 percent ofthe total protein in the refractile particle preparation, and theidentity of this protein as human growth hormone was verified by Westernblot. The yield of refractile particles was about 10-20 mg per gram ofwet cell paste.

FIG. 4A shows the refractile hGH containing bodies in a suspension ofthe pellet from the first spin.

FIG. 4B shows the results of SDS PAGE performed on killed (with acid)and unkilled cells from this preparation. The band corresponding to hGHin pellet from killed cells is enhanced.

EXAMPLE 3 Bovine Growth Hormone (bGH)

Recombinant DNA E. coli K12 carrying bovine growth hormone gene (strainW3110/pBGH-1) as described in U.S. application Ser. No. 303,687 filedSept. 18, 1981, now abandoned, was grown in fermenter and harvested, asdescribed in Example 1. (The cells were treated with 0.25 percent phenoland 0.25 percent toluene in the fermenter before harvest.) Refractileparticles were isolated and showed a single protein band in SDS-PAGEcorresponding to a standard at 22,000 daltons molecular weight. Adensitometer scan of the gel showed this band was over 90 percent of thetotal protein in the particles. After dissolving the particles in 7Mguanidine and dialysis in 7M urea, the presence of bGH was verified byradioimmunoassay (RIA). The yield of refractile particles was about 20mg per gram of wet cell paste.

EXAMPLE 4 Porcine Growth Hormone (pGH)

Recombinant DNA E. coli K12 carrying porcine growth hormone gene (strainW3110/pGH-exl) as described in U.S. application Ser. No. 439,977, filedNov. 8, 1982, was grown in fermenter and harvested as described inExample 1. (The cells were treated with 0.25 percent phenol and 0.25percent toluene in the fermenter before harvest.) Refractile particleswere isolated showed a single protein band in SDS-PAGE corresponding toa standard at 22,000 daltons molecular weight. A densitometer scan ofthe gel showed this band was over 90 percent of the total protein loadedon the gel. After dissolving the particles in 7M guanidine and dialysisin 7M urea, the presence of pGH was verified by radioimmunoassay (RIA).The yield of refractile particles was about 20 mg per gram of wet cellpaste. FIG. 4A shows the purity and concentration of refractile bodiescontaining pGH in the cell paste suspended in buffer, FIG. 4B shows thispaste after sonication and FIG. 4C after low-speed spin

EXAMPLE 5 Human Fibroblast Interferon (FIF)

Recombinant DNA E. coli K12 carrying human fibroblast interferon gene(strain W3110/pFIF 347) as described in Shepard, M., et al, DNA, 1:125(1982) was grown in fermenter and harvested, and refractile particleswere isolated by the procedure described in Example 1. 2-mercaptoethanolSDS-PAGE of the resulting refractile particle preparation showed a majorband corresponding to 17,000 daltons molecular weight, representing 50percent of the total protein in the refractile particle preparation.Western blot showed the refractile protein reacted specifically toantibodies against pure human fibroblast interferon. The yield ofrefractile particles was about 10-20 mg per gram of wet cell paste.

EXAMPLE 6 Human Immune Interferon (IIF)

Recombinant DNA E. coli K12 carrying human immune interferon gene(strain W3110/pIFN-γ trp48) described in U.S. application Ser. No.312,489 filed Oct. 19, 1981, now abandoned, was grown in fermenter andharvested; refractile particles were isolated by the procedure describedin Example 1. 2-mercaptoethanol SDS-PAGE of the resulting refractileparticle preparations showed a major band corresponding to 17,000 daltonmolecular weight, representing 50 percent of the total protein in therefractile particle preparation. Western blot showed the refractileprotein reacted specifically to antibodies against pure human immuneinterferon. The yield of refractile particles was about 10-20 mg pergram of wet cell paste.

EXAMPLE 7 Tissue Plasminogen Activator (TPA)

Recombinant DNA E. coli K12 carrying human tissue plasminogen activatorgene, (strain 3110, pEPAtrp12) as described in U.S. application Ser. No.398,003 filed July 14, 1982 (abandoned in favor of continuation-in-partSer. No. 483,052 filed Apr. 7, 1983), was grown in a fermenter, cellsharvested, and the refractile particles were isolated from cell pasteaccording to the procedure of Example 1.

80-90 percent of the protein present in the refractile particlepreparation was TPA as measured by densitometry of β-mercaptoethanolSDS-PAGE and Western blot. The yield of TPA refractile particles wasabout 10-20 mg per gram of wet cell paste.

EXAMPLE 8 FMD Coat Protein

Recombinant DNA E. coli K12 carrying various strains of foot and mouthdisease virus antigen (W3110/pFMG, W3110/pFMB [A24], W3110/FMD [C3],W3110/FMC [A27]) as described in U.S. application Ser. No. 374,855 filedMay 4, 1982, now abandoned, were grown and harvested, and the refractileparticles isolated according to the procedure described in Example 1.

About 50 percent of the protein present in the refractile particlepreparations were cloned FMD coat protein gene products in each casemeasured by densitometry of 2-mercaptoethanol SDS-PAGE and Western blot.

EXAMPLE 9 Refractile Protein Enhancement by Cell Killing Step

The amount of human growth hormone (hGH) in the cytoplasm vs the amountin refractile bodies was determined by a comparison study on killed andnonkilled cells: E. coli K-12 cells which were capable of expressing hGH(strain W3110/p107) (see Example 2) were grown and harvested bycentrifugation as set forth in paragraph A of Example 1. Prior tocentrifugation, the medium was divided and one portion was treated with0.25 percent phenol and 0.25 percent toluene and incubated at 37° C. forfour hours. The cells which were treated by killing were designated "PT"cells and those which were not killed "NPT" cells.

A. Extracts of Total Cellular Protein with SDS

Equal samples of the PT and NPT cells were subjected to identicaltreatment by disrupting each of the cell samples by sonication in asolution comprising 5 ml of 50 mM Tris containing 10 mM EDTA plus 250 ulof 20 percent SDS. The suspensions were vortexed for 0.5 minute, andthen centrifuged. The supernatants were assayed for hGH byradioimmunoassay (RIA) and by SDS PAGE. Both PT and NPT cells showedsubstantially identical activities in RIA, specifically 8.3×10⁶ and8.7×10⁶ units per ml, respectively. Presumably, the SDS extractionprocedure recovers both initially soluble and initially insoluble humangrowth hormone. The conforming SDS-PAGE results are shown in FIG. 6, aswell as the results of SDS-PAGE performed on the samples treated asdescribed in the next paragraph.

B. Experimental Extracts

Two further equal samples of PT and NPT cells were treated identicallyby extraction into 5 ml each of 50 mM Tris containing 10 mM EDTA, withvortexing for 0.5 minutes. The suspensions were then spun down atapproximately 10,000 xg for 10 min i.e. a low speed spin (LSS) and thesupernatant and pellet assayed separately. Presumably, the pelletprotein will be insoluble in the absence of SDS. The NPT cells showedactivity in the supernatant at a level of 4.6×10⁶ units per ml (about1/2 that of the SDS extract), but greatly diminished activity (0.42×10⁴units per ml) in the pellet. The PT cells, on the other hand, showed agreat reduction in hGH activity in the supernatant compared to the SDSextract, as measured by RIA--0.26×10⁴ units per ml, and also, littleactivity in the pellet as solubilization of the pellet having beeneffected. Similar results were obtained if samples were treatedcorrespondingly and subjected to higher speed centrifugation (HSS) i.e.35,000×G for 30 minutes.

C. Comparison of Results

FIG. 6 summarizes the results using SDS PAGE. SDS extracted cells ofcourse, showed equivalent intensities in the band corresponding to hGH.NPT cells showed substantial amounts of the band corresponding to hGH inboth the supernatant and the pellet for both high speed and low speedcentrifugation separations. On the other hand, the PT cells showed adiminution both in low speed and high speed treatments of the amounts ofhGH in the supernatant, but enhanced amounts in the pellet.

EXAMPLE 10 Capsid Protein of FMD Virus A Multistep Process

E. coli K12 harboring a gene encoding FMD virus type A24 (W3110/pFMB[A24] as described in U.S. application Ser. No. 374,855 filed May 4,1982, was grown to a cell density corresponding to about 30-50 O.D.units at 550 mm; or 40 g wet cell paste per liter whole broth in M9 orsimilar salts medium plus tryptophan at about 40-120 mg/l and glucose atno more than about 5 percent wt/vol of medium. See Experiments inMolecular Genetics. (J. H. Miller, Cold Spring Harbor Laboratory, N.Y.(1972).) The culture (10 liters) was brought to 0.25 percent in each ofphenol and toluene for at least 1/2 hour, and then centrifuged. The cellpaste was frozen for convenient storage prior to protein purification.The purification as performed in this Example is outlined in Scheme 3:##STR3##

Just before use, the cell paste was thawed in a refrigerator. 500 g ofthe thawed paste was dispersed in 5 liters Buffer A (50 mM phosphate, 5mM EDTA, 500 mM NaCl, 15 mM β-mercaptoethanol (BME), pH 7). A TekmarDispax model SD-45 with a G-450 generator (3 min. at full speed) wasused to obtain a uniform suspension.

The suspension was then homogenized with a Manton-Gaulin homogenizer(Type 15 M) run at 6000 psi for 2 passes, with cooling between cycles,and the homogenate centrifuged in a Beckman RC3 at 5000 rpm for 30minutes at 4° C. SDS-PAGE showed the supernatant to containsubstantially the same mixture of proteins as the uncentrifugedManton-Gaulin homogenate, but to have a greatly diminished bandcorresponding to that of the FMD protein.

The pellet contained approximately one half of the initial cell pastemass. The supernatant was decanted and discarded. The pellet was washedby dispersing in fresh buffer A (2 liter/200 g pellet) using the DispaxSD-45, and the suspension centrifuged again in the RC3 at 5000 rpm for30 minutes at 4° C. The supernatant was decanted and discarded, thepellet (137 gm) showed an enhanced band corresponding to FMD protein inSDS-PAGE, representing approximately 50 percent of the protein in thepellet.

The pellet was then extracted for FMD protein by suspending in 1 literof Buffer B (50 mM phosphate, 1 mM EDTA, 15 mM BME, 7M guanidinehydrochloride, (GuHCl), pH 7.0) using the Dispax SD-45. The suspensionwas stirred overnight, and clarified by spinning in a Sorvall SS-34rotor at 19,000 rpm for 3.0 hours at 4° C.

The pellet was discarded, and the FMD protein solution chromatographedon Sephacryl S-300 (Pharmacia). The gel was first equilibrated in BufferB and packed in a 5×50 cm column according to manufacturer'srecommendation. The void volume fractions (beginning at 270 ml) wereturbid; even though the solution that was applied to the column was not.The FMD protein-containing fractions emerged at 450-650 ml and wereclear. The FMD protein content was verified by dialyzing aliquots of thecolumn fractions against 8M urea and analyzing by SDS-PAGE. (Guanidineprecipitates with SDS and must be removed).

The FMD-protein containing fractions were pooled and dialyzed againstfour changes of a 20-fold excess of Buffer C (14 mM Tris 15 mM BME, 8Murea, pH 8.3) at 4° C. (The 8M urea substitutes for GuHCl in keeping theFMD protein in solution).

An aliquot of retentate was brought to pH 10 with NaOH and passed over aDE52 column (1.0 cm×19 cm) equilibrated with Buffer C adjusted to pH 10with NaOH. The FMD protein was not retained by the resin, whereas themajority of the E. coli contaminants were adsorbed. The protein in theflowthrough volume was 96 percent FMD protein as found by SDS-PAGE andrepresents a yield of 30 g/kg cell paste used or about 90 percent. Thismaterial may be used as final product if the presence of urea isacceptable in the administration of active material.

In the present preparation, the urea was removed without precipitationof the FMD protein by dialysis using 4 mg/ml protein against a 250-foldvolume excess of 1M urea, 14 mM Tris, 0.1 percent BME, pH 7 at 4° C.,followed by dialysis of the retentate against water. A slight cloudinessformed which was removed by centrifugation. Precipitation of FMD proteinin water or buffer was apparently prevented by the gradual diminution inurea concentration. The FMD protein obtained was biologically active asshown by reactivity with FMD antiserum, Western blot, and by in vivoassays in cattle wherein immune response was elicited.

EXAMPLE 11 Purification of Porcine Growth Hormone (pGH) A MultistepProcess

E. coli K12 (W3110/pPGH-exl) (Example 4) were grown as in Example 1 to30-50 O.D. units at 550 nm; 40 g wet cell paste per liter. The culturewas brought to 0.25 percent in both phenol and toluene for 1/2 hour. Thebroth was then centrifuged and the cell paste frozen for storage.

Just before use, 384 g of the cell paste was thawed in a refrigerator,dispersed in 3.9 liters cold Buffer A (50 mM phosphate, 5 mM EDTA, 500mM NaCl, pH 7). A Tekmar Dispax model SD-45 with a G-450 generator (3min. at setting of 60) was used to obtain a uniform suspension, and thesuspension was homogenized with a Manton-Gaulin homogenizer (Type 15 M)run at 6500 psi for 2 passes, with cooling between cycles.

The homogenate (4.3 liters) was centrifuged in a Sorvall RC3 at 5000 rpmfor 35 minutes at 4° C. The supernatant was decanted and shown bySDS-PAGE to contain substantially the same mixture of proteins as thehomogenate before centrifuging, but with a greatly diminished bandcorresponding to pGH.

The supernatant was discarded and the pellet was dispersed in freshBuffer (A (1.5 liter/150 g pellet) using the Dispax SDS-45. Thesuspension was centrifuged again in the RC3 at 5000 rpm for 35 minutesat 4° C. The supernatant was decanted off and discarded.

The pellet (106 g) was dissolved in 0.750 l of Buffer B (50 mM Tris, 1mM EDTA, 100 mM BME, 7M guanidine hydrochloride, pH 8.8) using theDispax SD-45 and then stirred overnight. The cloudy solution wasclarified by spinning in a Sorvall SS-34 rotor at 19,000 rpm for 6.0hours at 4° C., and the pellet was discarded.

690 ml of the supernatant (total volume 740 ml) was chromatographed atroom temperature on a 10×85 cm Sephacryl S-300 (Pharmacia) column in twoaliquots (350 ml and 345 ml). The column was equilibrated and run with7M guanidine HCl, 50 mM Tris, 1 mM EDTA, 50 mM BME, pH 8.9. Aliquots ofthe column fractions were dialyzed against 8M urea 0.1 percent BME, 25mM Tris, pH 7, and analyzed by SDS-PAGE, to determine the presence ofpGH.

The pGH-containing fractions were pooled and dialyzed against fourchanges of an approximately 20-fold excess of 15 mM Tris HCl, 50 mM BME,7M urea, pH 9.0 at 4° C. The retentate (3900 ml) was brought to pH 7.0with HCl and passed over a DE52 column (10 cm×11.5 cm) equilibrated with15 mM Tris, 7.5M urea, 50 mM BME, pH 7.0 at room temperature. The columnwas flushed with 1 column volume equilibrating buffer to wash outentrained pGH.

The DE52 pool (4000 ml) was titrated to pH 10 with NaOH, and dialyzed inSpectrapor 1 against three 100-liter changes of 25 mM Tris, 1 percentmannitol, pH 10 at 4° C. The retentate was pooled, and diluted to 10.4liters with dialysate, sterile filtered, and lyophilized. Ten g ofbiologically active protein (by Western blot) of 98 percent purity inpGH was obtained.

EXAMPLE 12 Refolding of Prorennin using Sulfitolysis

432 grams of cell paste from E. coli K-12 (strain W3110/pRIAX, whichcarries the gene encoding for prorennin as described in copendingapplication Ser. No. 501,351 filed June 6, 1983 were suspended in 3liters of buffer comprising 50 mM Tris HCl, 5 mM EDTA, pH 7.5. Thesuspension was subjected to cellular disruption by a Manton Gaulin pressat 6,000 psi for four passes. The disrupted suspension was thencentrifuged for 30 minutes at 4,000 xg and the supernatant removed anddiscarded. The pellet was resuspended in 2 liters of the same buffer aswas used in the original suspension and again centrifuged for 30 minutesat 4,000 x g. The supernatant was again discarded and the pelletdissolved in 6M guanidine hydrochloride containing 50 mM Tris at pH 8.The solubilized refractile protein was then sulfonated by bringing theguanidine solution to 20 mg/ml in sodium sulfite and 10 mg/ml in sodiumtetrathionate by adding an aliquot of a clarified stock solution freshlyprepared of these components. The sulfonation was allowed to proceed forfour hours at room temperature.

The solution was then diafiltered into 5M urea containing 50 mM TrisHCl, pH 7.5 for five hours. The diafiltered solution was then placed ona 10×35 cm column of DE52 which had been washed with the same "binding"buffer solution (5M urea, 50 mM Tris HCl, pH 7.5). The solution wasloaded at 750 ml per hour and washed overnight with the binding buffer.

The DE52 column was then eluted using a 0 to 0.15M NaCl gradient with aflow rate of 1 liter per hour over a 16 hour period. The majority of theprotein eluted at approximately 0.070M NaCl. The prorennin containingfractions were again diafiltered against 5M urea, 50 mM Tris HCl pH 8.5and the solution brought to 1 mM in GSH and 0.1 mM in GSSG and allowedto incubate at room temperature overnight. An additional diafiltrationto remove urea was conducted against 50 mM Tris, pH 8.0 containing 0.1mM GSH. The resulting solution, free of denaturant, contained 5.5 gramsof protein, or approximately 1 percent yield overall. The protein showedbiological activity by reaction against prorennin antiserum and byactivity (after autocatalytic activation) in a standard milk clottingassay.

EXAMPLE 13 Preparation of Active Urokinase Through Partial Sulfitolysis

Urokinase containing refractile bodies were isolated from E. coli K12(strain W3110/pUK 33trpLE_(L)) as described in U.S. application Ser. No.368,773 filed Apr. 15, 1982 (abandoned in favor of continuation-in-partSer. No. 474,930 filed Mar. 14, 1983) by the procedure set forth inExample 1. The refractile bodies were dissolved in 5M guanidine HCl,containing 50 mM Tris, pH 8.0. The solution was brought to 0.2 mg per mlin sodium sulfite and 0.1 mg per ml sodium tetrathionate, and incubatedovernight at room temperature.

The solution was then diluted to a level of 1.5M guanidine HCl with pH9.0, 50 mM Tris buffer, and brought to 10 mM GSH: 1 mM GSSG. The dilutedguanidine solution containing the dissolved protein was then againincubated overnight at room temperature and dialyzed into aqueoussolution. While the refractile bodies showed activity in a standardbioassay for urokinase of 0.25 PU/mg, the urokinase resulting from theprocedure herein set forth gave an activity of 150 PU/mg.

EXAMPLE 14 Reactivation of Urokinase by Redox Buffer Refolding

Refractile bodies prepared as set forth in Example 13 were dissolved in5 M guanidine hydrochloride in 50 mM Tris, pH 8.0, and then the solutiondialyzed into 2M urea, 50 Tris hydrochloride pH 7. The solution was thenbrought to 10 mM GSH:1 mM GSSG and incubated overnight at roomtemperature. The resulting solution containing refolded protein was thendialyzed into aqueous medium. The resulting solution contained urokinasewhich showed 30 PU/mg activity.

EXAMPLE 15 Refolding of Sarc Protein

Sarc, a protein originally isolated from sarcoma tumors was obtained byapplying the procedures of Example 1 to transformed cells preparedaccording to the procedure set forth in McGrath, J. P. and Levinson, A.D., Nature, 295: 423 (1982). To 3 mg of this protein which was insolubleunder native buffer conditions, was added 3 ml 7M guanidine, 300 μl 1MTris, pH 8, 20 μl 0.5M EDTA and 400 μl of a solution containing 200 mgper ml sodium sulfite and 100 mg per ml sodium tetrathionate. Thesolution was let stand at room temperature overnight and remained acloudy suspension.

The suspension was dialyzed against 7M urea containing 5 mM Tris, pH 8.To half of this solution was added 300 μl 0.1M glycine, pH 9.5 and 90 μl10 mM β-mercaptoethanol; and the solution allowed to stand overnight.After dialysis against 50 mM Tris, pH 8.5, the same protein was solubleand was capable when injected into mice of inducing antibodiesprecipitable against authentic sarc protein.

EXAMPLE 16 Solution in Guanidine and Extraction into Urea

E. coli K/2 (W3110/pFMB [A24]) (Example 8) was grown as described inParagraph A of Example 1 and harvested by centrifugation. The cell paste(281 g wet weight) was suspended in 10 volumes phosphate extractionbuffer (PEB: 50 mM NaH₂ PO₄, 5 mM EDTA, 0.5M NaCl, 0.1 percent BME, pH7.0) with an ultratorex. The resulting suspension was passed through aprechilled Manton Gaulin twice at 600 psi and 1 liter/minute. Aftercentrifugation at 5000 rpm (1/2 hour) in RC5B centrifuge the pellet wasresuspended in 10 volumes PEB with an ultratorex (10 min.) Thesuspension was centrifuged at 5000 rpm in RC5B centrifuge for 1/2 hourand the resulting pellet taken up in 20 volumes Urea-Tris buffer (UTB:8M urea (freshly prepared and deionized), 0.14M Tris, 0.1 percent BME,pH 8.3) and heated for 1/2 hour in a boiling water bath. After coolingto room temperature, 4 volumes of acetone were added and the solutionwas stirred at ˜6° C. for 11/2 hours. The suspension was thencentrifuged at 5000 rpm in the RC3B and the resulting pellet taken up in10 volumes PEB. After heating the suspension for 1/2 hour on a boilingwater bath the material was recentrifuged in RC5B at 5000 rpm and theresulting pellet resuspended for 48 hours in 2.2 liters 7 M GuHCl, 0.1percent BME.

The sample was then dialyzed versus 3 changes of UTB at pH 8.0 and thenchromatographed on a DE-52 cellulose column (5×8 cm) equilibrated withUTB, pH 8.0. The column wash was collected as two fractions, a clear andturbid fraction. The clear fraction (>90 percent VP3) was concentratedto ˜1.3 mg/ml and tested for biological activity.

A control batch of anitigen was prepared using E. coli K12 (W3110/pFMB[A24]) grown, harvested, and treated as above, but wherein UTB wassubstituted for GuHCl in taking up the pellet.

Samples were tested by injecting into guinea pigs and obtaining theantiserum after 28 days. The antiserum was then tested for antibodytiter by ability to protect suckling mice from FMD virus. The resultsare expressed as the negative log of the dilution of antiserum capableof conferring immunity on 50 percent of the mice (-log PD₅₀).

Antisera from injection of 100 μg protein of the above preparation had-log PD₅₀ values of 3.2-3.4, while antisera from injections of 100 μgprotein of extracts made from the controls had log PD₅₀ values less than0.3.

I claim:
 1. A process for re-activating proteins formed as refractilebodies in host cells, said proteins not subjected to CNBR treatmentwhich process comprises:(a) converting said proteins not subjected toCNBR treatment and dissolved in a strongly denaturing solution to thecorresponding protein-S-sulfonate; (b) converting the stronglydenaturing solution to a weakly denaturing solution in which theprotein-S-sulfonate remains dissolved; and (c) treating the resultingprotein-S-sulfonate with a sulfhydryl containing moiety containing asmall percentage of its disulfide.
 2. The process of claim 1 wherein theweakly denaturing solution is 1-9M in urea.
 3. The process of claim 1wherein the strongly denaturing solution is 4 to 9M in a guanidine salt,and the weakly denaturing solution is 1 to 9M in urea or 0.5 to 2M inguanidine hydrochloride.
 4. The process of claim 1 wherein thesulfhydryl containing moiety is reduced glutathione.
 5. The process ofclaim 4 whwerein the dissolved protein is converted to theprotein-S-sulfonate in (a) by treating the solution with a mixture of asulfite and a weak oxidizing agent.
 6. The process of claim 1 whereinthe sulfite is sodium sulfite and the weak oxidizing agent is sodiumtetrathionate.
 7. A process for re-activating heterologous proteinsformed as refractile bodies in host cells said proteins not subjected toCNBR treatment which process comprises:(a) converting said proteins notsubjected to CNBR treatment and dissolved in a strongly denaturingsolution to the corresponding protein-S-sulfonate; (b) replacing thestrongly denaturing solution with a weakly denaturing solution; (c)subjecting the dissolved protein-S-sulfonate to one or more purificationprocedures selected from the group consisting of ion exchangechromatography and gel permeation chromatography; and (d) treating thepurified protein-S-sulfonate from (c) with a sulfhydryl reagentcontaining a small percentage of its disulfide form.
 8. The process ofclaim 7 wherein the strongly denaturing solution is 4 to 9M in aguanidine salt and the weakly denaturing solution is 1 to 9M in urea or0.5 to 2M in guanidine hydrochloride.
 9. The process of claim 7 whereinthe dissolved protein is converted to the protein-S-sulfonate in step(a) by treating the solution with a mixture of sulfite and weakoxidizing agent.
 10. The process of claim 7 which includes carrying outa gel permeation chromatography step in (c).
 11. The process of claim 7which includes carrying out ion exchange chromatography in step (c). 12.The method of claim 7 wherein the purification procedure includes theuse of ion exchange chromatography comprising DEAE cellulose.
 13. Themethod of claim 12 wherein the DEAE cellulose absorbs contaminants ofthe heterologous protein, but not the heterologous protein.
 14. Proteinreactivated by the process of claim
 1. 15. Protein reactivated by theprocess of claim
 7. 16. The process of claim 1 including removing anyremaining denaturant from the solution.
 17. The process of claim 7including removing substantially all of the remaining denaturant.